Nucleic acid detection sensor

ABSTRACT

A nucleic acid detection sensor comprises a plurality of nucleic acid chain fixed electrodes to which a probe nucleic acid chain is fixed, and a counter electrode which is arranged opposite to the nucleic acid chain fixed electrode, and a current flowing between the counter electrode and the nucleic acid chain fixed electrode.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 09/961,249 filed Sep. 25, 2001, now U.S. Pat. No. 6,818,109 andbased upon and claims the benefit of priority under 35 U.S.C. § 119 toJapanese Patent Application No. 2000-301516, filed Sep. 29, 2000, theentire contents each of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a nucleic acid detection sensor whichelectrochemically detects whether a target nucleic acid chain in a testliquid has a specific base sequence.

2. Description of the Related Art

Recently, a genetic test technique with a nucleic acid chain fixed array(DNA array) is made attention as a nucleic acid detection sensor (see“Beattie et al. 1993, Fodor et al. 1991, Khrapko et al. 1989, Southernet al. 1994”).

DNA array indicates the array of a glass and a silicon with several cmsquares where DNAs with different 10¹ to 10⁵ kinds of arrays is fixed.The signed test liquid gene is reacted with a fluorescent dye and aradioisotope (RI), etc. on the array, or the unsigned test liquid geneand the compound of the sign oligonucleotide are reacted by the sandwichhybridization. When a complementary array to the DNA on the array existsin the test liquid, the signal (fluorescent intensity and RI intensity)which is derived from the sign by a specific part on the array isobtained. If the arrangement and the position of the fixed DNA are knownbeforehand, the base sequence which exists in the test liquid gene canbe easily checked. Since a lot of information on the base sequence withthe small amount sample can be obtained, DNA array is expected very muchnot only in the gene detection technique and also in the sequencetechnical (see “Pease et al. 1994, Parinov et al. 1996”).

There are a fluorescent detection method, RI intensity detection method,and an electrochemical detection method, etc. as a technique whichdetects the nucleic acid which is combined with the nucleic aciddetection sensor. A sign of the sample gene and a complex system are notrequired in an electrochemical technique. Therefore, the miniaturizationof the system can be expected. In addition, since the electrode used,the electrochemical technique has an advantage that an electric reactivecontrol can be easily performed.

Especially, among the nucleic acid detection sensors which use anelectrochemical technique, the sensor having a DNA array configurationin which a plurality of electrodes where a different probe nucleic acidchain is fixed are arranged in a X-Y matrix is expected as an extremelyuseful technical which can detect many kinds of nucleic acids with ashort time. However, the equal voltage should be applied to a lot ofnucleic acid chain fixed electrodes in this sensor. Therefore, thissensor has the problems such as the circuit configuration is complex,and the response speed and accuracy are not sufficient.

BRIEF SUMMARY OF THE INVENTION

An object of the present invention is to provide a nucleic aciddetection sensor which can detect many kinds of nucleic acids withhigh-speed and high-accuracy.

A first nucleic acid detection sensor according to the present inventionis characterized by comprising: a plurality of nucleic acid chain fixedelectrodes to which a probe nucleic acid chain is fixed; and a counterelectrode which is arranged opposite to the nucleic acid chain fixedelectrode, wherein a current flowing between the counter electrode andthe nucleic acid chain fixed electrode. Since the nucleic acid chainfixed electrode and the counter electrode are opposing arranged, themeasurement of high accuracy can be promptly performed by the decreasedamount of the test liquid.

A second nucleic acid detection sensor according to the presentinvention by comprising: a plurality of nucleic acid chain fixedelectrodes to which the probe nucleic acid chain is fixed; a counterelectrode, a current flowing between each of the nucleic acid chainfixed electrodes and the counter electrode; and a reference electrodeprovided for each of the nucleic acid chain fixed electrodes, configuredto make a voltage between the nucleic acid chain fixed electrode and thecounter electrode constant. The measurement sensitivity is improvedsince the reference electrode is arranged for each nucleic acid chainfixed electrode.

A the third nucleic acid detection sensor according to the presentinvention by comprising: a plurality of nucleic acid chain fixedelectrode, to which a probe nucleic acid chain is fixed, arranged in amatrix; a plurality of scanning lines configured to select the pluralityof nucleic acid chain fixed electrodes one by one; a plurality of signallines configured to transmit a measurement signal from the plurality ofnucleic acid chain fixed electrodes; a plurality of switching elementsconnected with the plurality of signal lines; and an A/D converterconnected with the plurality of switching elements. The configurationbecomes simple since only one A/D converter is prepared because theoutput line of the signal is shared with the switching element.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is a schematic diagram which shows a nucleic acid detection chipaccording to the embodiment of the present invention in which aplurality of electrodes are arranged;

FIG. 2 is a figure, which shows an arrangement of an electrode in anucleic acid detection chip according to the embodiment of the presentinvention and the signal line;

FIG. 3 is a figure, which shows an another arrangement of an electrodein a nucleic acid detection chip according to the embodiment of thepresent invention and the signal line;

FIG. 4 is a figure, which shows an another arrangement of an electrodein a nucleic acid detection chip according to the embodiment of thepresent invention and the signal line;

FIG. 5 is an expansion figure of a unit division of a nucleic aciddetection chip according to the embodiment of the present inventionwhere the plurality of electrode is arranged;

FIG. 6 is an expansion figure of a unit division of a nucleic aciddetection chip according to the embodiment of the present inventionwhere the plurality of electrode is arranged;

FIG. 7 is an expansion figure of a unit division of a nucleic aciddetection chip according to the embodiment of the present inventionwhere the plurality of electrode is arranged;

FIG. 8A to FIG. 8C are figures which show the nucleic acid detectionchip which can be installed in the nucleic acid detection system;

FIG. 9A and FIG. 9B are figures which show a nucleic acid detection chipwhich can be attached to the nucleic acid detection system;

FIG. 10 is a figure, which shows a nucleic acid detection system towhich the nucleic acid detection chips are arranged;

FIG. 11A and FIG. 11B are figures which show the nucleic acid detectionchip according to the embodiment of the present invention stored in thecontainer;

FIG. 12 is a figure, which shows the substrate to which the nucleic aciddetection chip according to the embodiment of the present inventionshould be installed;

FIG. 13A and FIG. 13B are figures which show a nucleic acid detectionchip according to the embodiment of the present invention stored in thecontainer;

FIG. 14A to FIG. 14D are figures which compares a nucleic acid detectionchip according to the embodiment of the present invention in whichelectrodes are arranged at opposed positions and a conventional nucleicacid detection chip in which electrodes are not arranged at opposedpositions;

FIG. 15 is a figure, which shows an example of the circuit applied tothe second nucleic acid detection chip according to the embodiment ofthe present invention;

FIG. 16 is a figure, which shows another example of the circuit appliedto the second nucleic acid detection chip according to the embodiment ofthe present invention;

FIG. 17 is a figure, which shows another example of the circuit appliedto the second nucleic acid detection chip according to the embodiment ofthe present invention;

FIG. 18 is a figure, which shows the configuration of the nucleic aciddetection chip according to the embodiment of the present invention inwhich electrodes are arranged at the opposite positions;

FIG. 19 is a figure, which shows a wiring in the nucleic acid detectionchip according to the embodiment of the present invention in whichelectrodes are arranged at the opposite positions;

FIG. 20 is a figure, which shows the signal and the output signal toapply the voltage to each unit division; and

FIG. 21 is a figure, which shows a wiring in a nucleic acid detectionchip according to the embodiment of the present invention in whichelectrodes are arranged at the opposite positions.

DETAILED DESCRIPTION OF THE INVENTION

The nucleic acid detection sensor according to embodiment of presentinvention is characterized by comprising each of the followingconfigurations.

(1) The nucleic acid chain fixed electrode and the counter electrode areoppositely arranged.

(2) The reference electrodes are arranged for each cell.

(3) The output line of the signal is shared with the switching element.

In the specification, in the nucleic acid detection sensor according tothe present invention to which a plurality of nucleic acid chain fixedelectrodes is arranged, a “nucleic acid detection cell” means the unitdivision (unit cell) which comprises a couple of a nucleic acid chainfixed electrode and a counter electrode.

The probe nucleic acid chain is fixed to hybridize the target nucleicacid chain in the test liquid to the nucleic acid chain fixed electrode.The nucleic acid chain fixed electrode functions as an action electrodein the nucleic acid detection sensor according to the present invention.A “probe nucleic acid chain” indicates the nucleic acid chain fixed(combined) to the nucleic acid chain fixed electrode. A “target nucleicacid chain” is a nucleic acid chain which has a complementary basesequence for said probe nucleic acid chain and reacts the hybridizationwith said probe nucleic acid chain, and means the nucleic acid chainincluded in the test liquid.

The counter electrode functions as an auxiliary electrode to flow thecurrent which flows between nucleic acid chain fixed electrodes. Inaddition, the measurement method of the nucleic acid detection cell ofthe nucleic acid detection sensor according to the present invention maybe duplex system which uses the nucleic acid chain fixed electrode andthe counter electrode, applies an arbitrary voltage between the nucleicacid chain fixed electrode and the counter electrode, and detects anelectrochemical change generated between both electrodes. Themeasurement thereof may be an electrochemical measurement using triodesystem to connect a reference electrode with the nucleic acid chainfixed electrode and the counter electrode of the duplex system. Sincethe current flows to the counter electrode in the duplex system, thereis a disadvantage that the concentration of the carrier in theelectrode/interface, which determines the potential of the counterelectrode, changes and the potential, which becomes a reference,changes. On the other hand, in triode system, the potential (potentialof the reference electrode) which becomes a reference does not change,since the current flows between the nucleic acid chain fixed electrodeand the counter electrode, the current hardly flows between the nucleicacid chain fixed electrode and the reference electrode and between thecounter electrode and the reference electrode, and the voltage isapplied between the nucleic acid chain fixed electrode and the counterelectrode so that the desired potential may be applied to the referenceelectrode.

The finding of the target nucleic acid chain or the probe nucleic acidchain is obtained with the nucleic acid detection sensor according tothe present invention as follows. The voltage is applied between thenucleic acid chain fixed electrode and the counter electrode in thenucleic acid detection cell in the existence of the test liquidincluding the nucleic acid chain. After the hybridization is occurredbetween the target nucleic acid chain and the probe nucleic acid chain,an electrochemical change generated between electrodes is detected. Anelectrochemical change is generated between electrodes if the targetnucleic acid chain is hybridized with the probe nucleic acid. Therefore,if the corresponding change is detected whether the probe nucleic acidchain or the target nucleic acid chain has a specific base sequence canbe detected. It is preferable that an electrochemical change generatedbetween electrodes because of the above-mentioned hybridization is acurrent change which adds two chain recognition bodies in the testliquid, and derives from the two chemical change of the chainrecognition body. As a result, the measurement can be performed withease and high accuracy.

The nucleic acid chain having a known base sequence is used as a probenucleic acid chain to be fixed to the nucleic acid chain fixedelectrode. Whether the target nucleic acid chain, which reacts thehybridization in the test liquid with said probe nucleic acid chainexists may be detected. The nucleic acid chain with an unknown basesequence as a probe nucleic acid chain to be fixed to the nucleic acidchain fixed electrode is used. Whether or not the target nucleic acidchain which reacts the hybridization in test liquid with said probenucleic acid chain exists is detected by containing the nucleic acidchain having a-known base sequence in test liquid. Thus, the finding ofthe array of the probe nucleic acid chain with the unknown base sequencecan be obtained.

Typically, a different kind of the probe nucleic acid chain is fixed toeach of the plurality of nucleic acid chain fixed electrodes. To supplydifferent samples in each cell and perform the check of several samplesat a time, the same kind of probe nucleic acid chain may be fixed.

Since one nucleic acid chain fixed electrode is arranged to the eachnucleic acid detection cell, the finding of the array of the targetnucleic acid chain or the probe nucleic acid chain is obtained. Bychecking the target nucleic acid chain hybridize to the nucleic aciddetection cell. Therefore, it is preferable to arrange a switchingcircuit, a decoder circuit, or, a timing circuit to apply the electricsignal to each nucleic acid chain fixed electrode of each cell, acircuit which outputs an electric signal from each nucleic acid chainfixed electrode, and a switching circuit to output an electric signalfrom each nucleic acid chain fixed electrode to an external device tooperate the each nucleic acid detection cell independently.

The plurality of scanning lines are connected with the circuit such asthe switching circuits to apply an electric signal to each nucleic acidchain fixed electrode of each cell. The signal to turn on thetransistor, desirably, a switching element such as a thin filmtransistor, arranged between the nucleic acid chain fixed electrode andthe signal line is applied to the scanning line. A “signal line” means aconductor line which transfers the signal specifying an electric changefrom the nucleic acid chain fixed electrode. The nucleic acid chainfixed electrode is an action electrode in this specification. Thevoltage is applied to the nucleic acid chain fixed electrode when theswitching element is turned on according to the signal from the scanningline, then an electrochemical change is occurred. The change in thevoltage (and/or current) by the electrochemical change is transmitted bythe signal line. It is preferable to use the matrix system used todisplay the liquid crystal in the control of the plurality ofelectrodes, particular. Furthermore, it is preferable that there is anactive matrix system to use the MOSFET. The scanning circuit of the MOSimage sensor type can be used.

FIG. 1 shows the structure of the typical nucleic acid detection sensorwhich comprises the circuit to apply the voltage to each nucleic acidchain fixed electrode. FIG. 1 shows a case of measurement system of atwo-electrode system. The switching element 103 connected with eachnucleic acid chain fixed electrode 102 turns off and on by the signalbeing output to the scanning line driving circuit 107 in order to drivethe scanning line 104 from the timing circuit 106 one by one in thenucleic acid detection sensor of FIG. 1. The counter electrode isconnected with the power supply (not shown in the figure) through thepontiostat circuit 110. The voltage is applied to the nucleic acid chainfixed electrode 102 and counter electrode 101 when the switching element103 opens and closes one by one. As a result, the nucleic acid (notshown in the figure) which hybridizes to the nucleic acid chain fixedelectrode 102 can be detected electrochemically. An electrochemicalchange is transferred to the signal detection circuit 109 through thesignal line 105 and is detected.

The signal line is preferably coated with the insulation materialexcluding the contact with the switching element as shown in FIG. 2.FIG. 2 is side figure when the cell (rectangle with dotted line) for thenucleic acid detection in the nucleic acid detection sensor of FIG. 1 iscut along parallel to the scanning line to cross the nucleic acid chainfixed electrode and the counter electrode. In FIG. 2, the signal line202 coated by the insulation film 203, the nucleic acid chain fixedelectrode 204, and the counter electrode 205 are arranged on theinsulation substrate 201. Since the signal line 202 is soaked in thetest liquid, the signal line 202 is coated by the insulation film 203excluding the contact of the signal line 202 and the switching element(not shown in the figure: contact).

The arrangement of the signal line coated with the insulation material,the switching element, and the electrode is not limited to thearrangement of FIG. 2, but may be arbitrary arrangement. FIG. 3 is anexample of such an arrangement, and switching elements 304 and 305 arerespectively put under the nucleic acid chain fixed electrode 302 andthe counter electrode 303 (or, reference electrode) arranged on theinsulation substrate 301. The switching elements 304 and 305 are coatedby the insulation films 306 and 307 which exist respectively on bothsides thereof. Since the contact part of the signal line (not shown inthe figure) and the switching element is not contacted with the testliquid 308 even when the test liquid 308 is added on the nucleic acidchain fixed electrode and the counter electrode if the switching elementis put under each electrode as shown in FIG. 3, it is excellent ininsulation properties. The arrangement of FIG. 4 also has the excellentinsulation properties, since the switching element is put under eachelectrode as well as FIG. 3. But, the point in which the switchingelement is exposed to the rear surface of the substrate differs from thearrangement of FIG. 3.

It is preferable that each electrode which constructs the nucleic aciddetection cell is formed on the insulation substrate. As a material ofthe insulation substrate, for example, inorganic insulation materialssuch as glass, fused silica, silicon, alumina, sapphire, forsterite, andsilicon carbide, silicon oxide, and silicon nitride, or, organicmaterials such as polyethylene, ethylene, polypropylene,polyisobutylene, polyethylene terephthalate, no saturation polyester,include fluorine resin, polyvinyl chloride, polyvinylidene chloride,polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin,polyacrylonitrile, polystyrene, acetal, polycarbonate, polyamide, phenolresin, urea resin, epoxy resin, and melamine resin, styreneacrylonitrile copolymer, acrylonitrile butadiene styrene copolymer,silicon resin, polyphenylene oxide, and polysulfone can be used, but thematerial is not limited to these materials.

It is preferable that each electrode and the circuit, etc. are separatedby the insulation material. The insulation material used by the presentinvention is not limited, but it is preferable to use photo polymer, anda photoresist material. The photoresist for an optical exposure, thephotoresist for the far ultraviolet, and the photoresist for X-ray andthe photoresist for the electron beam are used as a resist material. Thephotoresist for an optical exposure includes cyclized rubber, thepolycinnamic acid, and the novolac resin as a main raw material.Cyclized rubber, phenol resin, polymethyl isopropenyl ketone (PMIPK),and polymethyl methacrylate (PMMA), etc. are used for the photoresistfor the far ultraviolet. The material of the description in the “ThinFilm Handbook” (Ohmsha, Ltd.) in addition to COP and the metal acrylatecan be used for resist for X-ray. In addition, the material of thedescription can be used for the above-mentioned document such as PMMA inresist for the electron beam. It is preferable that the resist used hereis 1 mm or less and more than 100 Å. It becomes possible to make thearea constant by covering the electrode with the photoresist, andperforming the lithography. As a result, the fixed amount of the probenucleic acid chain becomes uniform between each electrode, and anexcellent measurement in reproducibility is enabled. Conventionally, itis general to remove the resist material finally, but the resistmaterial may be used as a part of the electrode without removing in thenucleic acid chain fixed electrode. In this case, it is necessary to usea high water-proof material for the resist material to be used. Amaterial excluding the photoresist material may be used as theinsulation layer formed on the upper portion of the electrode. Forexample, oxide, nitride, carbide, and other alloy of Si, Ti, Al, Zn, Pb,Cd, W, and Mo, Cr, Ta, and Ni, etc. may be used. After forming a thinfilm by using a sputter, an evaporation or a CVD etc. from thesematerials, the electrode exposure part is patterned by the photolithography and the area is constantly controlled.

Preferably, 10¹ to 10⁵ nucleic acid chain fixed electrodes are arrangedon the insulation substrate. The material of a desirable nucleic acidchain fixed electrode is gold, and other materials can be used, forexample, metal single substance such as alloy of gold, silver, platinum,mercury, nickel, palladium, silicon, germanium, gallium, and tungstenand alloy thereof, or, carbon etc. such as graphite and glassycarbon,or, oxide and compound thereof may be used. These electrodes can bemanufactured by a plating, a print, a sputter, and an evaporation, etc.When the evaporation is performed, the electrode film can be formed withthe resistive heating method, the high frequency heating method, and theelectron beam heating method. The electrode film can be formed by a DCtwo poles sputtering, a bias sputtering, an asymmetric AC sputtering, agettering sputtering, and a high frequency sputtering when thesputtering is used. Here, when the gold is used for the electrode, theorientation index in a (111) plane of the crystal structure of the goldis important. The orientation index is obtained from the followingequations by the method of Willson.Orientation index (hkl) =IF _((hkl)) /IFR _((hkl))

Hkl; plane index

IF_((hkl)); Relative intensity in (hkl) plane

IFR_((hkl)); IF_((hkl)) as standard gold described in ASTM card

Here, in case of the nucleic acid chain fixed electrode for the nucleicacid chain detection, it is required that the orientation index is oneor more, in addition, it is preferable that the orientation index is twoor more. It is also valid to heat the substrate to improve theorientation at the evaporation or the sputtering. It is preferable thatthe heating temperature is a range of 50° C. to 500° C., though theheating temperature is not limited. It becomes possible to uniformlycontrol the nucleic acid chain fixed amount by controlling theorientation. When the above-mentioned electrode material of the goldetc. are evaporated or sputtered to the substrate such as the glass, itbecomes possible to form a stable electrode layer by existing titaniumor chrome, copper, nickel, and alloy thereof solely or by combiningbetween the substrate and the gold as a bonding layer.

The shape of the nucleic acid chain fixed electrode is not the limited,and the shapes shown in FIG. 5 to FIG. 7 are preferable. It isadvantageous since the contact area of the nucleic acid fixed electrodeand the counter electrode (or, reference electrode) is large if theshapes of FIG. 6 and FIG. 7 are used. FIG. 5 to FIG. 7 are figures whichexpand the nucleic acid detection cell (rectangle of dotted line in FIG.1). As well as FIG. 1, the nucleic acid chain fixed electrodes 501, 601and 701 are connected with the signal lines 505, 605 and 705 through theswitching elements 503, 603 and 703, respectively. The counterelectrodes (or, reference electrode) 502, 602 and 702 is arranged in theneighborhood of the nucleic acid chain fixed electrodes 501, 601 and701.

It is preferable to activate the surface of the electrode to fix theprobe nucleic acid chain to the nucleic acid chain fixed electrode.Activation can be performed by the potential sweep in the sulfuric acidsolution. Activation can be performed even by the mixed acid and theaqua regia, etc. The material which constructs the probe nucleic acidchain is not limited, but DNA, RNA, PNA, and other nucleic acids analogcan be used.

The method of fixing the probe nucleic acid chain is not especiallylimited. For example, it is possible to perform fixation easily by usingthe bond of the thiol radical and gold which are introduced into theprobe nucleic acid chain. Additionally, it is possible to performfixation by physical adsorption, chemical adsorption, the canal bond,embedding, and the covalent bond, etc. The condensation medicines suchas the biotin-avidin bonds and carbodiimides can be used. Fixation canbe facilitated by modifying the surface of the electrode with themolecule having the functional group beforehand in these cases. Inaddition, to control non-peculiar adsorption of a nucleic acid to thesurface of the electrode and the intorcalators etc., it is preferable tocover the surface of the electrode with the mercaptan such asmercaptoethanols and lipids such as stearylamides.

Hereinafter, the method of fixing the probe nucleic acid chain to thenucleic acid chain fixed electrode which consists of gold will bedescribed as an example. After washing by the demineralized water, theelectrode performs the activation processing. The sulfuric acid solutionof 0.1 to 10 mmol/L is used for activation. Potential is scanned in thissolution within the range of 1 v/s to 100,000 v/s within the range of−0.5 to 2V (vs Ag/AgCl). As a result, the surface of the electrode isactivated to the state that the probe nucleic acid chain can be fixed.The thiol radical is introduced to 5′ or 3′ end of the probe nucleicacid chain used for fixation. The probe nucleic acid chain made a thioldissolves to the solution of the reducer such as DTT immediately beforefixation, and removes DTT by the extraction operation with the gelfiltration or the acetic acid ethyl etc. immediately before use.Fixation is very easy. The probe nucleic acid chain is dissolved in thebuffer liquid within the range of pH5 to pH10 as becoming within therange of 1 ng/mL to 1 mg/mL within the range of ion intensity of 0.01 to5. The activated electrode is soaked. The reaction of fixation isperformed for about ten minutes to around one evening within the rangeof 4 to 100° C.

It is preferable to store the electrode after fixing the probe nucleicacid chain in the condition where the nucleic acid decomposition ferment(nuclease) does not exist, and by shading if possible. However, when itis short-term, it is possible to be stored in the wet condition. It ispreferable that the composition of the medium is a composition of theliquid to which the hybridization reacts, Tris-EDTA buffer liquid, ordemineralized water. It is preferable that the storage temperature is 4°C. or less, preferably, −20° C. When the nucleic acid chain fixedelectrode to which the probe nucleic acid chain is fixed is stored at along term, it is preferable to store in a dry condition. It is possibleto perform dry with freeze-drying and air drying, etc., though themethod of keeping to dry is not especially limited. It is preferablethat it is an inert gas, nitrogen, dry air such as argon or a vacuum,though gas phase of dry is not especially limited.

The operativeness of the test can be raised by putting the sign or thebar code on each electrode.

The probe nucleic acid chain can be fixed comparatively easily with adevice of fixation which is called DNA spotter and DNA arrayer at thetime of fixing the probe nucleic acid chain on the electrode. In thiscase, it is preferable to use the spotter of an ink jet system or anelectrostatic system to prevent the surface of the electrode from beingdamaged. It is also possible to synthesize the nucleic acid chaindirectly on the surface of the electrode.

One or more counter electrodes are arranged on the nucleic aciddetection sensor according to the present invention. When a singlecounter electrode is arranged, the plurality of nucleic acid chain fixedelectrodes commonly use a single counter electrode.

If the desired voltage can be applied to the nucleic acid chain fixedelectrode, the distance of the nucleic acid chain fixed electrode andthe counter electrode is not especially limited. It is preferable toarrange the distance within 1 cm, for example, to make the responsespeed fast.

It is desirable to arrange the counter electrode to become an equaldistance from all the nucleic acid chain fixed electrodes in order toapply an equal voltage to all the nucleic acid chain fixed electrodes.

The material is not especially limited to the material used for thecounter electrode, and the material used by the nucleic acid chain fixedelectrode can be used.

When the nucleic acid detection sensor according to the presentinvention is measured by the triode system, the reference electrode isarranged. The silver/silver chloide electrode, mercury/mercury chlorideelectrodes, etc. can be used as a reference electrode, but otherarbitrary electrodes can be used.

It is general to arrange the counter electrode or the referenceelectrode in the same substrate as the nucleic acid chain fixedelectrode, but the counter electrode or the reference electrode may bearranged in the part excluding this.

Since the measurement accuracy is improved, it is desirable that a shapeof reference electrode is a shape of enlarging the surface area andpreventing the flow of test liquid from obstructing, though the shapethereof is not especially limited. For example, electrodes suit such acondition by making the reference electrode and the nucleic acid chainfixed electrode a comb shape in which electrodes thereof are engagedmutually.

It is preferable that the nucleic acid detection sensor according to thepresent invention configures the nucleic acid detection system. Thecorresponding system has a basic configuration comprising one or aplurality of substrates on which the plurality of nucleic acid detectioncells are formed, the container which is an airtight container to holdthe substrate and has at least one or more openings to transfer theliquid and a space for storing the liquid, and a terminal to connectwith an external equipment.

On the system, it is preferable to comprise a circuit to apply anelectric signal to the counter electrode and the nucleic acid chainfixed electrode, a switching circuit to apply an electric signal to eachcounter electrode and each nucleic acid chain fixed electrode, a circuitwhich outputs an electric signal from each nucleic acid chain fixedelectrode, a switching circuit to output an electric signal from eachnucleic acid chain fixed electrode outside, a power supply, apotentiostat, and a the waveform generation device. To the system, it ispreferable to integrate circuits such as a decoder circuit to output anelectric signal to the MOSFET switching element at specific positionarranged in matrix and a nucleic acid chain fixed electrode, a switchingcircuit, a timing circuit, memory, an A/D converter, a waveformgeneration device, a power supply, a potentiostat, and an electricsignal detection circuit on one sensor.

A nucleic acid extraction mechanism, a nucleic acid refinementmechanism, and a nucleic acid amplification mechanism, etc. can beintegrated on the nucleic acid detection system. If the nucleic aciddetection system comprising these mechanisms is used, all series ofoperations of the extraction, amplification, and the detection, etc. ofa nucleic acid can be automatically performed.

In FIGS. 8A to 8C show an example of the nucleic acid detection sensorwhich can construct the nucleic acid detection system.

The nucleic acid detection sensor of FIGS. 8A to 8C comprises aplurality of scanning lines 801, signal lines 802 arranged to cross thescanning lines 801, switching elements 803 such as thin film transistorsarranged to each intersection in the scanning lines 801 and the signallines 802, nucleic acid chain fixed electrodes 804 connected with theswitching elements 803, a scanning line driving circuit 805 to driveeach scanning lines 801, a first substrate 807 (FIG. 8A) on which asignal line driving circuit 806 to drive each signal line 802 isarranged, and a second substrate 809 (FIG. 8C) on which each counterelectrode 808 is arranged. Each counter electrode 808 is connected withthe potentiostat (not shown in the figure). Though only one nucleic acidchain fixed electrode is written in FIG. 8, actually, one nucleic acidchain fixed electrode is arranged respectively on the rectangular ofeach inside of the surround to two adjacent scanning line and twoadjacent signal lines.

In order to detect the target nucleic acid in the test liquid, thedriving signal is transmitted from the scanning line driving circuit 805to the switching element 803, after the test liquid is injected into thespace which lies between the first substrate 807 and the secondsubstrates 809. The switching element 803 is turned on by the drivingsignal output from the scanning line driving circuit 805, and thenucleic acid chain fixed electrode 804 and signal line 802 areelectrically connected. The voltage is applied between the nucleic acidchain fixed electrode 804 and the counter electrode 808 when the nucleicacid chain fixed electrode 804 and signal line 802 are electricallyconnected. As a result, for example, the stuff such as the intorcalatorsinserted in the target nucleic acid hybridized to the nucleic acid chainfixed electrode 804 is oxidized. The current generated by the oxidationflows to the pad 810 provided to one end of the signal line 802 throughthe signal line 802, and is detected and measured with an externalequipment for the current detection connected with the pad 810.

In the nucleic acid detection sensor of FIG. 8, a nucleic acid detectionpart 811, a scanning line driving circuit 805, and a signal line drivingcircuit 806 are formed on the first substrate 807 integrally, and thenucleic acid detection sensor is installed to the nucleic acid detectionsystem comprising the signal detection part and is used.

In the shape shown in FIG. 9A and FIG. 9B, the counter electrode 905 maybe connected with scanning lines 901 electrically and may be arranged inthe vicinity of the nucleic acid chain fixed electrode. In FIG. 9A andFIG. 9B, the counter electrode 905 has a comb shape each of whosebranches are branched to three, and is arranged so as to mutually engagewith the nucleic acid chain fixed electrode 905 having the same shapethereof.

Though the nucleic acid detection sensor to which the referenceelectrode is not provided is shown in FIG. 8A, FIG. 8B, FIG. 9A, andFIG. 9B, it is preferable to provide the reference electrode. Thereference electrode may be a comb electrode which is mutually engaged tothe nucleic acid chain fixed electrode, and can be god combination,float, and be type electrode in each other as shown in FIGS. 9A and 9B.

An example of the circuit of the embodiment to which the referenceelectrode is provided in each nucleic acid chain fixed electrode will bedescribed later.

The outline of the nucleic acid detection system to which the nucleicacid detection sensor according to the present invention is arranged isshown in FIG. 10.

The nucleic acid detection System 1007 shown in FIG. 10 comprises anucleic acid detection sensor 1001, a nucleic acid detection sensorfixation device 1002, an electric signal measurement device 1003, a CPU1004, a power supply 1005, and, a display device 1006.

The nucleic acid detection sensor is usually attachably and detachablyarranged on the substrate 1102 with the connection terminal 1101, andstored in the container 1108 as shown in FIG. 11 in the above-mentionedsystem. The substrate 1102 has a connection terminal insertion part 1201in the surroundings thereof, for example, as shown in FIG. 12. In FIG.11, the test liquid 1103 is injected from the test liquid injectionentrance 1105 provided to the bottom to soak the nucleic acid detectionsensor 1104 in a state of closing the test liquid outlet 1106. After thenucleic acid detection sensor 1104 is soaked with the test liquid 1103,the nucleic acid included in the test liquid 1103 is hybridized with thenucleic acid chain fixed electrode (not shown in the figure) on thenucleic acid detection sensor 1104. When the nucleic acid detectionsensor 1104 is humidified while hybridizing, the evaporated test liquidis exhausted through an air hole 1107. The target nucleic acid ishybridized with the nucleic acid chain fixed electrode (not shown in thefigure) on the nucleic acid detection sensor 1104 if the target nucleicacid is included in test liquid. Therefore, after the test liquid 1103is exhausted from the test liquid outlet 1106, the test liquid 1103keeps combining with the nucleic acid chain fixed electrode. The testliquid injection entrance 1305 and the test liquid outlet 1306 may beprovided at a vertical position of substrate 1302 as shown in FIG. 13.

Hereinafter, the operation to obtain the finding of the target nucleicacid chain in the test liquid or the probe nucleic acid chain with anucleic acid detection sensor according to the present invention will beexplained in detail.

First, the test liquid including the target nucleic acid chain isinjected to the space which lies between the nucleic acid chain fixedelectrode and the counter electrode.

The detected target nucleic acid chain is not especially limited, andmay be the nucleic acid chain of virus, bacillus, fungous, and helminthetc., a cause genes of an inherited disease, and a marker genes ofvarious diseases etc. For example, it is possible to be used to detectvirus infection syndromes such as hepatitis virus (A, B, C, D, E, F, andG type), HIV, influenza virus, herpes group virus, adenovirus, humanpolyoma virus, human papilloma virus, human parvovirus, mumps virus,human rotavirus, enterovirus, Japanese encephalitis virus, dengue fevervirus, rubella viruses, and HTLV, bacillus infection syndromes such asyellow staphylococcus, hemolytic streptococcus, escherichia coli,enteritis vibrio, helicobacter pylori, campylobacter, cholera bacterium,dysentery bacterium, salmonella, senior ell, gonococcus, and squirrelterrier bacterium, leptospire, legionalla bacterium, spirochete,pneumonia mycoplasma, rickettsia, chlamydia, malaria, dysentery amoebas,and causes of a disease fungous, and helminth, fungous, parasite orfungus. It can be also used for a check of inherited disease, retina budcell tumor, Wilm's tumor, family character large intestines polyposis,inherited non-polyposis colic cancer, neurofibroma, familial breastcancer, xeroderma pigmentosum, brain tumor, mouth cancer, gullet cancer,stomach cancer, and colic cancer, check of tumor disease such as livercancer, pancreatic cancer, lung cancer, goiter, mastadenoma, urinaryorgans tumor, virilia tumor, female genital tumor, ecphyma, bone-softpart tumor, leukemia, lymphoma, solid tumor, etc. It can be adopted toall fields to which the gene check is necessary in the food check,quarantine, medicine check, legal medicine, agriculture, stock raising,fishery, and forestry, etc. besides the medical treatment. In addition,the detection of restriction fragment length polymorphism (RFLP), singlenucleotide polymorphisms (SNPs), and the micro satellite array, etc. isalso possible. It is also possible to use for analyzing the unknown thebase sequence.

The test liquid which contains these target nucleic acids is notespecially limited, for example, blood, serum, white blood corpuscle,urine, service, semen, saliva, organization, culture cell, andexpectoration, etc. may be used. The nucleic acid component is usuallyextracted from these test liquids. The extraction method is notespecially limited, and may use liquid-liquid extraction methods ofphenol-chloroform method, etc. and the solid-liquid extraction methodswhich use carrier. It is also possible to use method QIAamp ofextracting a nucleic acid on the market (made by the QIAGEN company) andSumai-test (made by the Sumitomo Metal Industries, Ltd. company), etc.

The hybridization reaction is performed by the extracted nucleic acidcomponent and the nucleic acid chain detection electrode after the testliquid is injected into the space. A reactive solution is performed inthe buffer liquid within the range of ion intensity 0.01 to 5 and therange of pH5 to pH10. Sulfuric acid dextran, salmon sperm DNA,sweetbread DNA, EDTA, and the surface-active agent, etc. which are thehybridization promotion medicine in this solution can be added. Thenucleic acid component extracted here is added, and thermal deformed at90° C. or more. The insertion of the nucleic acid chain detectionelectrode can be performed immediately after deformation or after quickcooling to 0° C. A reactive speed can be improved by the operation suchas the stir or shaking while reacting. A reactive temperature is withinthe range of 10° C. to 90° C. and a reactive time is from one minute ormore to about one evening. The hybridization reaction can be controlledelectrochemically and necessary time of reaction can be shortened to afew minutes by applying for the plus potential to the nucleic acid chainfixed electrode, but it is necessary from several hours to several days,conventionally. On the other hand, nonspecific bond can be removed byapplying for minus potential to the surface of the electrode.

When the hybridization reaction ends, the nucleic acid chain fixedelectrode is washed. The buffer liquid within the range of pH5 to pH10and the ion intensity thereof is within the range of 0.01 to 5 is usedfor washing.

The duplex chain cognitive body, that is, an intercalator whichselectively combines with a duplex part (hybrid of the probe nucleicacid chain and the target nucleic acid chain) formed on the surface ofthe electrode after washing, reacts and an electro-chemical measurementis performed. The duplex chain cognitive body is a double strandednucleic acid recognizing substance which binds specifically to a doublestranded nucleic acid and is active physicochemically to the reactionsystem of said nucleic acid probe and said gene sample. The intercalatorused here is not limited, and bisintercalator, trisintercalator, andpolyintercalator, etc. such as Hoechst 33258, acridine orange,quinacrine, daunomycin, metalointercalator, and bisacridine can be usedas the intorcalator. It is also possible to use the living body highmolecules such as organic compounds such as metallic complex such asruthenium which is called metalointercalator, cobalt, and iron andethidium bromide, antibodies, and ferments.

The concentration of the intorcalator is different depending on thekind, but it is used within the range of generally 1 ng/mL to 1 mg/mL.In this case, the buffer liquid within the range of pH5 to 10 and theion intensity within the range of 0.001 to 5 is used.

The nucleic acid chain fixed electrode is washed after reacting with theintorcalator, and an electrochemical measurement is performed. Anelectrochemical measurement is performed by three electrode type, thatis, the reference electrode, the counter electrode and the actionelectrode or two electrode type, that is, the counter electrode and theaction electrode. In the measurement, a potential more than a potentialto which the intorcalator electrochemically reacts is applied, and thereactive current value which derives from the intorcalator is measured.In this case, the potential can be sweeped at a fixed velocity, or beapplied with the pulse or can be applied with fixed potential. Thecurrent and the voltage are controlled by using devices such aspotentiostat, digital multi meters, and the function generators for themeasurement. The concentration of the target gene is calculated fromcalibration curve based on the obtained current value.

An electrochemical signal can have the oxidation reduction currentchange, the oxidation reduction potential change, the electric capacitychange, the resist change, and the electrochemistry emission change tothe index. The effect of these signal change is promoted by using thematerial which combines with duplex chain nucleic acid such asintorcalator etc. specifically.

The first nucleic acid detection sensor according to the presentinvention is characterized by arranging the nucleic acid chain fixedelectrode and the counter electrode oppositely so that the test liquidmay flow between the nucleic acid chain fixed electrode and the counterelectrode.

FIG. 14C and FIG. 14D show the nucleic acid detection sensors whichconstruct the conventional DNA array. A plurality of nucleic acid chainfixed electrodes 1401, to which the probe nucleic acid chain 1402 withan already-known array is fixed, and the counter electrode 1404 arearranged on the same substrate 1403, and the test liquid 1406 flows onabove-mentioned substrate 1403. In the corresponding arrangement, thedistance of the counter electrode 1404 and each nucleic acid chain fixedelectrode 1402 is different in each nucleic acid fixed electrode 1401.In such a configuration, there is a case the distance of the nucleicacid chain fixed electrode 1401 and the counter electrode 1404 mightbecome far as shown in the left end of the figure, and the responsespeed becomes slow. Since the distance of the counter electrode 1404 andeach nucleic acid chain fixed electrode 1401 is different in eachnucleic acid chain fixed electrode 1401, enough measurement accuracycannot be achieved.

On the other hand, in the first nucleic acid detection sensor accordingto the present invention shown in FIG. 14A and FIG. 14B, the nucleicacid chain fixed electrode 1401 and the counter electrode 1404, to whichthe probe nucleic acid chain 1402 is fixed, are plate-like electrodes,and are arranged oppositely to grasp so that the test liquid. Accordingto the corresponding arrangement, all of each nucleic acid chain fixedelectrode 1401 on the first substrate 1403 can be arranged to an equaldistance from the counter electrode 1404 on the second substrate 1405and the neighborhood of the counter electrode 1404. Therefore, by usingthe nucleic acid detection sensor in which the electrodes are arrangedin such a manner, it becomes possible to apply an equal voltage to allthe target nucleic acids in the test liquid 1406, which is hybridizedwith the probe nucleic acid chain 1402 on each nucleic acid chain fixedelectrode 1401, to be detected. Therefore, the measurement accuracy andthe response speed improve. Since the test liquid 1306 is injectedbetween the first substrate 1303 and the second substrates 1305, theamount of a necessary test liquid can be decreased. FIG. 14A shows thenucleic acid detection sensor that the reference electrode 1407 is notarranged on the first substrate 1403. FIG. 14B shows the nucleic aciddetection sensor that the reference electrode 1407 is arranged on thefirst substrate 1403.

In the first nucleic acid detection sensor according to the presentinvention, when the nucleic acid chain fixed electrode is formed on theinsulation substrate, the counter electrode is arranged to place a flowpath of the test liquid with the nucleic acid chain fixed electrode, andis arranged in a substrate different from the substrate where thenucleic acid chain fixed electrode.

The position of the counter electrode and the nucleic acid chain fixedelectrode is not especially limited. It is desirable to arrange thecounter electrode to become an equal distance from all the nucleic acidchain fixed electrodes to apply an equal electrode to all the nucleicacid chain fixed electrodes, though both electrodes are arranged on adifferent substrate. For example, when the nucleic acid chain fixedelectrode is arranged on a plane, the counter electrode may be arrangedon a plane parallel to the plane. When the nucleic acid chain fixedelectrode is arranged on the sphere, the counter electrode may bearranges on the concentric sphere of the sphere.

In the first nucleic acid detection sensor, it is preferable the both ofthe nucleic acid fixed electrode and the counter electrode have flatsurfaces and the flat surfaces are arranged oppositely.

The first nucleic acid detection sensor according to the presentinvention comprises a plurality of nucleic acid detection cells, and oneor more nucleic acid chain fixed electrodes is arranged in each cell.The counter electrode may be provided for each nucleic acid chain fixedelectrode. The counter electrode may be common among the plurality ofnucleic acid detection cells and may be one, for example, in a word, forthe plurality of nucleic acid chain fixed electrodes.

The material of the counter electrode which should be arranged in thefirst nucleic acid detection sensor according to the present inventionand the distance with the nucleic acid chain fixed electrode are theabove-mentioned.

It is general to arrange the reference electrode in the same substrateas the nucleic acid fixed electrode when the reference electrode isarranged in the first nucleic acid detection sensor according to thepresent invention. The reference electrode may be arranged in the partexcluding this.

The second nucleic acid detection sensor according to the presentinvention is characterized by comprising a reference electrode in eachcell.

In the second nucleic acid detection sensor according to the presentinvention, the counter electrode may be common to the plurality ofnucleic acid chain fixed electrode or may be arranged for each nucleicacid detection cell. The counter electrode may be connected with eitherof the signal line or scanning line when the plural connect counterelectrodes are arranged.

The material of the nucleic acid chain fixed electrode, the counterelectrode, and the reference electrode and the operation etc. for themeasurement of nucleic acids are as being described to a generalconfiguration of the nucleic acid detection sensor according to thepresent invention mentioned above and directions. That is, by using theelectrochemical reaction by the hybrid nucleic acid chain formed betweenthe probe nucleic acid chain and the target nucleic acid chain, whetheror not the probe nucleic acid chain or the target nucleic acid chain hasa specific base sequence is detected.

Thus, the measurement accuracy improves by decreasing the uncompensatoryresistance between the nucleic acid chain fixed electrode and thereference electrode if the reference electrode is provided to eachnucleic acid chain fixed electrode. It is preferable to comprise thereference electrode to each nucleic acid chain fixed electrode tocontrol the potential of each nucleic acid chain fixed electrode.

The second nucleic acid detection sensor according to the presentinvention uses, for example, the potentiostat circuit for the minutecurrent measurement comprising an operational amplifier 1607 whichfunctions as a control amplifier, a voltage floor amplifier, and acurrent floor amplifier, an operational amplifier 1608, and anoperational amplifier 1609 as shown in FIG. 15. The plurality of nucleicacid chain fixed electrodes are actually arranged to the second nucleicacid detection sensor according to the present invention though only onenucleic acid chain fixed electrode is shown in the potentiostat circuitof FIG. 15 for easiness.

This circuit comprises three operational amplifiers with the function ofeach control amplifier, voltage flower amplifier, and the current flooramplifier. These circuits are different from the conventional circuit inthe point for the minute current measurement. Therefore, thepotentiostat circuit used in the nucleic acid detection sensor may be acircuit for the minute current measurement.

The function of each operational amplifier in the circuit of FIG. 15 isas follows.

The operational amplifier 1607 configures a part of the inversionamplifier, and by applying the voltage of (1+Zf/Rf) times of ef (ef isassumed to mean the potential of point f when the common potential isassumed to be reference here, and it is same as follows) to the counterelectrode 1602 ef is kept constant for ea (That is, Vcc) (Here, Zf showselectrochemical impedance from counter electrode 1602 to the referenceelectrode 1603). Since the operational amplifier has the negativefeedback, ea is equal to eb (potential of common). In the figure, thoughthe common is grounded, it is not unnecessary to ground.

The operational amplifier 1608 has a function to amplify an input powerto Zin/Zout times (Zin and Zout are the input impedance and outputimpedances, respectively). Since Zin is very high compared with Zout,the output power becomes remarkably large compared with the input power.Internal resistance of the reference electrode 1603 can be disregardedby the function of the operational amplifier 1608.

Since the operational amplifier 1609 has the negative feedback, eg isequal to eh, therefore, when the nucleic acid chain fixed electrode 1601is connected with the signal with the switching element 1604, thepotential of the nucleic acid chain fixed electrode 1601 becomes equalwith the potential of common. Therefore, the operational amplifier 1609operates to keep the potential of the nucleic acid chain fixed electrode1601 which is the action electrode to the common potential. If the ratioof the resistance (not shown in the figure) between point 0 and point aand the resistance between point a and point f is set to 1 when theinput voltage is assumed to be V, the potential of the referenceelectrode becomes −V by the action of the operational amplifier 1603.The resistance of resistor in the circuit and the presence of the use ofthe resistance may be properly selected according to the desiredamplification rate etc. Since the potential of the nucleic acid chainfixed electrode 1601 is equal to the potential of common, an equalvoltage to the input voltage is accurately applied between the nucleicacid chain fixed electrode 1601 (action electrode) and the referenceelectrode 1603. The current caused by applying the voltage to thenucleic acid chain fixed electrode 1601 by the switching element 1604connected with the scanning line 1606 from point g on signal line 1605to point i through the resistor 1610 since the point g is groundedvirtually. The largeness of the current can be measured by measuring thevoltage down by the resistor 1610.

When the resistor 1610 is arranged between a point g and a point i, theerror is occurred in the potential of the nucleic acid chain fixedelectrode 1601 by the potential difference at both ends of the resistor.However, even if the resistor 1610 is arranged between the point g andthe point i, the error is not occurred in the potential of the nucleicacid chain fixed electrode 1601 since eg is kept the potential ofcommon. Therefore, it becomes possible to measure an electrochemicalmeasurement of high accuracy.

The circuit of FIG. 16 is another potentiostat circuit used for thesecond nucleic acid detection sensor, and has a function to keep thevoltage to be constant as well as the circuit of FIG. 15. Therefore, thefunctions of operational amplifiers 1707, 1708 and 1709 are the same asthe corresponding operational amplifiers shown in FIG. 15.

Since the reference electrode is arranged to each nucleic acid chainfixed electrode as well as FIG. 15, the sensor circuit for the nucleicacid detection of this embodiment has the measurement accuracy in thesensor for compared with the conventional circuit.

Though only one reference electrode is drawn in FIG. 16 for easiness,one or more reference electrodes are arranged to each nucleic acid chainfixed electrodes, actually.

Since the reference potential is held concurrently in the circuit ofFIG. 16 by the potential applied to scanning line, the wiring which isled from non- inversion input terminal of the operational amplifier 1708is used together to the plurality of electrode, and not included in thenumber of wirings for the nucleic acid detection cell.

As mentioned above, the nucleic acid detection sensor of this embodimentcan achieve very high measurement sensitivity with simple wiring.

The circuit of FIG. 17 is still another potentiostat circuit used forthe nucleic acid detection sensor of the second embodiment, and thecircuit of FIG. 17 has the function to keep the voltage to be constantas well as the circuits of FIG. 15 and FIG. 16. Therefore, details ofthe function of potentiostats 1807, 1808 and 1809 are as described inFIG. 15 or FIG. 16.

The circuit of FIG. 17 is different from the circuit of FIG. 16, and thereference electrode 1803 is connected with not the scanning line 1806but the signal line 1804. Therefore, the reference electrode 1803 is notconnected with the scanning line 1806 in the circuit of FIG. 17.Therefore, the reference potential of the reference electrode does nothold concurrently with the potential of the scanning line 1806, and theapplied potential can be freely set. Therefore, many kinds ofintorcalators can be used compared with the circuit of FIG. 16 in thecircuit of FIG. 17.

The switching element may be omitted though the reference electrode 1803is connected with switching element 1804 in FIG. 17.

The nucleic acid chain fixed electrode 1801 and the reference electrode1803 are placed both sides of the lead which are connected with thenon-inversion input terminal of the operational amplifier 1808 in FIG.17. The reference electrode 1803 may be arranged on the same side as thenucleic acid chain fixed electrode 1801 so that two electrodes arefaced.

As mentioned above, the circuit of FIG. 17 can achieve high measurementsensitivity, and can use many kinds of intorcalators.

The third nucleic acid detection sensor according to the presentinvention will be explained referring to FIG. 18 to FIG. 21. The thirdnucleic acid detection sensor according to the present invention ischaracterized in that the signal lines are shared by the switchingelement.

FIG. 18 is upper figure of the nucleic acid detection sensor which isusually used. In FIG. 18, the nucleic acid chain fixed electrodes 1901to which the probe nucleic acid chain (not shown in the figure) is fixedare arranged in the X-Y matrix of 4×3. The counter electrode is omittedin FIG. 18, though the counter electrode is located above the planewhere the nucleic acid chain fixed electrode 1901 is arrangedperpendicular thereto in the sensor for actual nucleic acid detection.

Each nucleic acid chain fixed electrode 1901 forms the nucleic aciddetection cell with the counter electrode.

Each nucleic acid chain fixed electrode 1901 is connected with thesignal line 1903 through the switching element 1902 such as transistors.The signal line 1903 is further connected with the amplifier 1904 andthe A/D converter 1905 to amplify the current from the nucleic acidchain fixed electrode 1901.

Since the clock signal is output from the timing pulse generator 1909 tothe switching element 1902 through the scanning line 1906, the nucleicacid chain fixed electrodes 1901 are scanned to become active from theleft end to an arrow direction in the figure one by one. The counter1908 and the X decoder 1907 of FIG. 18 control the ON-OFF on the signalline. When the nucleic acid chain fixed electrode 1901 becomes active,the voltage is applied between the nucleic acid chain fixed electrode1901 and the counter electrode (not shown in the figure) and theintorcalator inserted in the target nucleic acid which hybridizes withthe nucleic acid chain fixed electrode 1901 is oxidized. After anelectric change occurred at oxidation is amplified with said theamplifier 1904 through the signal line 1903, an electric change isA/D-converted with the A/D converter 1905.

FIG. 19 is a figure, which shows an example of the circuit of the thirdnucleic acid detection sensor according to the present invention. In thenucleic acid detection sensor of FIG. 19, the point, in which theswitching element is also arranged in a row direction and the scanningis performed from upper side to lower side of the FIG. 19, is differentfrom the nucleic acid detection sensor of FIG. 18.

In FIG. 19, the nucleic acid chain fixed electrode 2001 is arranged inthe X-Y matrix of 4×3, and the nucleic acid chain fixed electrode 2001and the counter electrode (not shown in the figure) construct thenucleic acid detection cell.

Each nucleic acid chain fixed electrode 2001 is connected with thesignal line 2004 through the amplifier 2002 and the electrode switchingelement 2003. The signal line switching element 2005 is connected with apart of each signal line 2004, and thereafter the signal lines 2004become one line, and is connected with the A/D converter 2006.

An electric signal is output from the column direction scanning circuit,which is constructed by the X decoder 2007 and the counter 2008, to theelectrode switching element 2003 one by one through the signal line2012. On the other hand, an electric signal is output from the rowdirection scanning circuit, which is constructed by the Y decoder 2009and the counter 2010 to the signal line switching element 2005 one byone.

If the clock signal generated from the timing pulse generator 2011 isoutput to the column direction scanning circuit and the row directionscanning circuit respectively as X direction clock signal and Ydirection clock signal as shown in FIG. 20, the voltage is applied tothe electrode from the first column and first row line (electrode in thetop of the left), the first column and second row line, the first columnand third row line, and the second column and first row line . . . oneby one. An electric change, which takes place because of the applicationfor the voltage is measured as a serial signal, and is A/D-convertedinto the output signal with the A/D converter.

In the nucleic acid detection sensor of FIG. 19, to detect an rowdirection electric signal one by one, the nucleic acid detection sensorusing the scanning circuit, which is constructed by the decoder and thecounter is shown. The decoder and the counter of FIG. 19 can be replacedwith the shift register circuit 2210 as shown in FIG. 21. Theconfiguration of the nucleic acid detection sensor of FIG. 21 is thesame as the nucleic acid detection sensor of FIG. 19 excluding decoderand the counter being replaced with the shift register circuit. Thus, ifthe shift register circuit is used, the external circuit configurationbecomes simple.

The third nucleic acid detection sensor shown in FIG. 19 and FIG. 21 hasthe effect of speeding up the measurement in compared with the nucleicacid detection sensor shown in FIG. 18.

It can be also possible to use the first to third nucleic acid detectionsensors according to the present invention alone, and can be used byproperly combining.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

1. A nucleic acid detection sensor comprising: a plurality of nucleicacid chain fixed electrodes to each of which a probe nucleic acid chainis fixed; a counter electrode which is arranged opposite to the nucleicacid chain fixed electrode, wherein a current flows between the counterelectrode and each nucleic acid chain fixed electrode; and a referenceelectrode, wherein the reference electrode and the nucleic acid chainfixed electrodes are formed on the same plane and the referenceelectrode is formed so as to surround the nucleic acid chain fixedelectrode.
 2. The nucleic acid detection sensor according to claim 1,wherein the counter electrode includes a plurality of counter electrodesand the plurality of counter electrodes are provided for the nucleicacid chain fixed electrodes, respectively.
 3. The nucleic acid detectionsensor according to claim 1, wherein each of the nucleic acid chainfixed electrodes has a flat plane to which the probe nucleic acid isfixed, the counter electrode has a flat plane, and the flat plane of oneof the nucleic acid chain fixed electrodes is arranged to face the flatplane of the counter electrode.
 4. The nucleic acid detection sensoraccording to claim 1, wherein the nucleic acid chain fixed electrodesand the counter electrode is arranged so that a test liquid can flowtherebetween.
 5. The nucleic acid detection sensor according to claim 1,wherein a test liquid is filled between the nucleic acid chain fixedelectrodes and the counter electrode so that a current change betweenthe nucleic acid chain fixed electrodes and the counter electrode causedby a hybridization of the probe nucleic acid and a nucleic acid in thetest liquid is detected.
 6. The nucleic acid detection sensor accordingto claim 1, wherein a duplex chain cognitive body is added to a testliquid filled between the nucleic acid chain fixed electrodes and thecounter electrode, and a current change between the nucleic acid chainfixed electrodes and the counter electrode caused by the duplex chaincognitive body is detected.
 7. The nucleic acid detection sensoraccording to claim 1, wherein the nucleic acid chain fixed electrodesare comb electrodes.
 8. The nucleic acid detection sensor according toclaim 1, further comprising a plurality of reference electrodes providedfor the nucleic acid chain fixed electrodes, respectively.
 9. Thenucleic acid detection sensor according to claim 8, wherein the counterelectrode includes a plurality of counter electrodes and the pluralityof counter electrodes are provided for the nucleic acid chain fixedelectrodes, respectively.
 10. The nucleic acid detection sensoraccording to claim 1, wherein the nucleic acid chain fixed electrodesand the reference electrode are comb electrodes, and the nucleic acidchain fixed electrodes and the reference electrode are arranged to bemutually engaged.
 11. A nucleic acid detection sensor comprising: aplurality of nucleic acid chain fixed electrodes to each of which aprobe nucleic acid chain is fixed; counter electrodes which are providedfor respective of the nucleic acid chain fixed electrodes, a currentflowing between each nucleic acid chain fixed electrodes and therespective counter electrode; and a plurality of reference electrodesprovided for respective of the nucleic acid chain fixed electrodes,wherein the nucleic acid chain fixed electrode are comb electrodes. 12.The nucleic acid detection sensor according to claim 11, wherein thereference electrode are comb electrode, the nucleic acid chain fixedelectrodes and the reference electrode are arranged to be mutuallyengaged.
 13. The nucleic acid detection sensor according to claim 11,wherein the counter electrode is comb electrode, the nucleic acid chainfixed electrodes and the counter electrode are arranged to be mutuallyengaged.
 14. The nucleic acid detection sensor according to claim 11,wherein the nucleic acid chain fixed electrodes and the counterelectrode are exposed to a test liquid, and a current change between thenucleic acid chain fixed electrodes and the counter electrode caused bya hybridization of the probe nucleic acid and a nucleic acid in the testliquid is detected.
 15. The nucleic acid detection sensor according toclaim 11, wherein a duplex chain cognitive body is added to the testliquid, and a current change between the nucleic acid chain fixedelectrodes and the counter electrode caused by the duplex chaincognitive body is detected.
 16. A nucleic acid detection sensorcomprising: a plurality of nucleic acid chain fixed electrodes to eachof which a probe nucleic acid chain is fixed; counter electrodes whichare provided for respective of the nucleic acid chain fixed electrodes,a current flowing between each nucleic acid chain fixed electrode andthe respective counter electrode; a plurality of reference electrodesprovided for respective of the nucleic acid chain fixed electrodes, afirst amplifier which inputs a signal from the reference electrode or ascanning line; a second amplifier configured to input a referencepotential to apply a predetermined potential to the counter electrode;and a reference resistor connected between an output side of the firstamplifier and the reference potential.
 17. The nucleic acid detectionsensor according to claim 16, wherein the nucleic acid chain fixedelectrodes and the counter electrode are exposed to a test liquid, and acurrent change between the nucleic acid chain fixed electrodes and thecounter electrode caused by a hybridization of the probe nucleic acidand a nucleic acid in the test liquid is detected.
 18. The nucleic aciddetection sensor according to claim 17, wherein a duplex chain cognitivebody is added to the test liquid, and a current change between thenucleic acid chain fixed electrodes and the counter electrode caused bythe duplex chain cognitive body is detected.
 19. A nucleic aciddetection sensor comprising: a plurality of nucleic acid chain fixedelectrodes to each of which a probe nucleic acid chain is fixed; acounter electrode, a current flowing between each of nucleic acid chainfixed electrodes and the counter electrode; a plurality of referenceelectrodes provided for the nucleic acid chain fixed electrodes,respectively, wherein the counter electrode and the nucleic acid chainfixed electrodes are formed on a same plane and the counter electrode isformed so as to surround the nucleic acid chain fixed electrodes, and aduplex chain cognitive body is added to the test liquid, and a currentchange between the nucleic acid chain fixed electrodes and the counterelectrode caused by the duplex chain cognitive body is detected.
 20. Thenucleic acid detection sensor according to claim 19, wherein the nucleicacid chain fixed electrodes and the counter electrode are exposed to atest liquid, and a current change between the nucleic acid chain fixedelectrodes and the counter electrode caused by a hybridization of theprobe nucleic acid and a nucleic acid in the test liquid is detected.21. The nucleic acid detection sensor according to claim 20, wherein aduplex chain cognitive body is added to the test liquid, and a currentchange between the nucleic acid chain fixed electrodes and the counterelectrode caused by the duplex chain cognitive body is detected.
 22. Anucleic acid detection sensor comprising: a plurality of nucleic acidchain fixed electrodes to each of which a probe nucleic acid chain isfixed; a plurality of counter electrodes provided for respective of thenucleic acid chain fixed electrodes, a current flowing between each ofthe nucleic acid chain fixed electrodes and the respective counterelectrode; and a plurality of reference electrodes provided forrespective of the nucleic acid chain fixed electrodes, wherein thereference electrode and the nucleic acid chain fixed electrodes areformed on a same plane and the reference electrode is formed so as tosurround the nucleic acid chain fixed electrode.
 23. The nucleic aciddetection sensor according to claim 22, wherein the nucleic acid chainfixed electrodes and the counter electrode are exposed to a test liquidand a current change between the nucleic acid chain fixed electrodes andthe counter electrode caused by a hybridization of the probe nucleicacid and a nucleic acid in the test liquid is detected.
 24. The nucleicacid detection sensor according to claim 23, wherein a duplex chaincognitive body is added to the test liquid, and a current change betweenthe nucleic acid chain fixed electrodes and the counter electrode causedby the duplex chain cognitive body is detected.